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Cited by 82Genomics and Chromatin DynamicsGene Regulatory Network AnalysisGenetic Associations and Epidemiology

Transcription factor binding site orientation and order are major drivers of gene regulatory activity

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Georgakopoulos-Soares, Ilias, Deng, Chengyu, Agarwal, Vikram, Chan, Candace S. Y., Zhao, Jingjing, Inoue, Fumitaka, Ahituv, Nadav·2023·Nature Communications
DOI 10.1038/s41467-023-37960-5PMC PMC10122648
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Abstract

The gene regulatory code and grammar remain largely unknown, precluding our ability to link phenotype to genotype in regulatory sequences. Here, using a massively parallel reporter assay (MPRA) of 209,440 sequences, we examine all possible pair and triplet combinations, permutations and orientations of eighteen liver-associated transcription factor binding sites (TFBS). We find that TFBS orientation and order have a major effect on gene regulatory activity. Corroborating these results with genomic analyses, we find clear human promoter TFBS orientation biases and similar TFBS orientation and order transcriptional effects in an MPRA that tested 164,307 liver candidate regulatory elements. Additionally, by adding TFBS orientation to a model that predicts expression from sequence we improve performance by 7.7%. Collectively, our results show that TFBS orientation and order have a significant effect on gene regulatory activity and need to be considered when analyzing the functional effect of variants on the activity of these sequences.

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  • The bottom-up synthetic approach can provide data of sufficient statistical power to characterize combinatorial action of TFBSs, as demonstrated by recent studies that have profiled tens of thousands to millions of synthetic or even random DNA sequences in cell lines or yeast.

    “and it can provide data of sufficient statistical power to characterize combinatorial action of TFBSs, as demonstrated by recent studies that have profiled tens of thousands to millions of synthetic or even random DNA sequences in cell lines or yeast.33,35,36”

    From Design principles of cell-state-specific enhancers in hematopoiesis (2025) · cites this paper as [33]

  • Measuring enhancer activity of synthetic, systematically designed DNA sequences with TFBSs placed in an otherwise inert sequence context is an alternative way to study the cis-regulatory code.

    “An alternative way to study the cis-regulatory code is to measure the enhancer activity of synthetic, systematically designed DNA sequences, where TF binding sites (TFBSs) are placed in an otherwise inert sequence context.27,30,31,32,33,34”

    From Design principles of cell-state-specific enhancers in hematopoiesis (2025) · cites this paper as [33]

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Cited by

Sorted by citations

Authors
Georgakopoulos-Soares, I., Deng, C., Agarwal, V., Chan, C. S. Y., Zhao, J., Inoue, F., & Ahituv, N.
Affiliations
  • University of California, San Francisco (US)
  • Kyoto University (JP)
Corresponding author
Ahituv, Nadav
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-04-22
Volume
14
Issue
1
Pages
2333-2333
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-37960-5
PMC
PMC10122648
OpenAlex
W4366769693
Cited by
82
References
6
Citations by year
2026: 31 · 2025: 25 · 2024: 24 · 2023: 2
FWCI
7.09
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
Funders
U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (1UM1HG011966)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Associations and Epidemiology
Keywords
transcription factor binding sites, gene expression regulation, promoter, massively parallel reporter assay, regulatory variants, transcriptional regulation
MeSH terms
Binding Sites, Gene Expression Regulation, Humans, Promoter Regions, Genetic, Protein Binding, Transcription Factors

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